Photochemically-induced crystallization of protein

I demonstrate photochemically induced crystallization of metastable hen egg-white lysozyme solution by weak UV irradiation for several tens seconds. The most effective irradiation time range is 10–60 s, and in this range the enzyme activity is maintained. Intermediates, neutral radicals at tryptophan residual produced by one-photon absorption, enhance nucleation. When the intermediate is selectively excited by visible light, the intermediate is denatured. At that time the light-induced nucleation is inhibited. This result indicates the intermediate induces nucleation. The radical forms lysozyme dimer that is detected by an SDS-PAGE electrophoresis experiment. An addition of polyethylene glycol (PEG) greatly enhances light-induced nucleation. PEG affects to shorten the intermediate radical lifetime, which suggests that PEG assists to form dimer. We consider that the photochemical dimer behaves as smallest cluster to grow critical nucleus. The smallest cluster formation is the rate determining step in classical nucleation theory due to surface energy disadvantage. The photochemical dimer is formed by a covalent bond, and the nucleation is initiated from stable dimer. The nucleation enhancement is reasonably explained. The present researches results point out the development of a new method for controlling nucleation and growth that could be applied for structural genomics and pharmaceutical industry for instance.     

Peptide mapping with mobile phases of intermediate pH value using capillary reversed-phase high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry

This investigation describes the separation of tryptic peptides by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) with eluents in the intermediate pH range, followed by in-line electrospray ionisation tandem mass spectrometry (ESI-MS/MS) analysis. For these purposes, gradient elution procedures with an aqueous eluent containing 20 mM ammonium formate, and an increasing content of acetonitrile or methanol, were employed. Compared to the analysis of the same tryptic peptides under low-pH conditions with an ion-pairing reagent, the increase in the pH with the 20 mM ammonium formate mobile phase led to significant changes in both peptide retention to the reversed-phase column and the collision-induced dissociation at the MS/MS stage as a consequence of the changes in the physico-chemical properties of these peptides, such as their overall charge, polarity and relative hydrophobicity. Thus, improved selectivity for the peptide separation and favourable tandem mass spectrometry analysis could be obtained with eluents in this intermediate pH range. The number of tryptic peptides identified by the new approach for the proteins investigated were significantly higher than that obtained by the conventional low-pH methods. Moreover, analysis of protein digests at very low concentrations was also performed under both acidic and intermediate pH conditions and similar improvements in selectivity and MS/MS detection limits were observed, i.e. identification of more distinct peptides and higher sequence coverage of the protein was obtained when eluents of intermediate pH were employed. This study therefore highlights the potential of conducting peptide mapping in the intermediate pH range to achieve more reliable and sensitive protein identifications with capillary RP-HPLC–ESI-MS/MS.     

The oxidation of formic acid at noble metal electrodes Part 4. Platinum + palladium alloys

Formic acid oxidation has been studied on Pt + Pd alloys of known surface composition using cyclic voltammetry. The results are interpreted in terms of a strongly bound (poisoning) intermediate which adsorbs only on the Pt atoms and a direct oxidation path which involves an intermediate occupying a single site and a second intermediate occupying two sites. Steady state oxidation currents on the alloys at potentials in the range of about 0.4 to 0.9 V are larger than expected from a linear combination of the currents on the pure metals. This effect may be due to the modification of metal properties at the junction between two types of metallic atoms.     

Intermediate Electrostatic Field for the Generalized Elongation Method

An intermediate electrostatic field is introduced to improve the accuracy of fragment‐based quantum‐chemical computational methods by including long‐range polarizations of biomolecules. The point charge distribution of the intermediate field is generated by a charge sensitivity analysis that is parameterized for five different population analyses, namely, atoms‐in‐molecules, Hirshfeld, Mulliken, natural orbital, and Voronoi population analysis. Two model systems are chosen to demonstrate the performance of the generalized elongation method (ELG) combined with the intermediate electrostatic field. The calculations are performed for the STO‐3G, 6‐31G, and 6‐31G(d) basis sets and compared with reference Hartree–Fock calculations. It is shown that the error in the total energy is reduced by one order of magnitude, independently of the population analyses used. This demonstrates the importance of long‐range polarization in electronic‐structure calculations by fragmentation techniques.     

Nucleophilic Substitution of Aliphatic Fluorides via Pseudohalide Intermediates

A method for aliphatic fluoride functionalization with a variety of nucleophiles has been reported. Carbon–fluoride bond cleavage is thermodynamically driven by the use of silylated pseudohalides TMS-OMs or TMS-NTf₂, resulting in the formation of TMS-F and a trapped aliphatic pseudohalide intermediate. The rate of fluoride/pseudohalide exchange and the stability of this intermediate are such that little rearrangement is observed for terminal fluoride positions in linear aliphatic fluorides. The ability to convert organofluoride positions into pseudohalide groups allows facile nucleophilic attack by a wide range of nucleophiles. The late introduction of the nucleophiles also allows for a wide range of functional-group tolerance in the coupling partners. Selective alkyl fluoride mesylation is observed in the presence of other alkyl halides, allowing for orthogonal synthetic strategies.     

Intramolecular vibrational redistribution in Ne-Br2: the signature of intermediate resonances in the excitation spectrum

Quantum-mechanical simulations of the Ne-Br(2)(B,v') excitation spectra produced after vibrational predissociation in the v'=20-35 range are reported. The aim is to investigate the signature in the excitation spectra of intermediate resonances lying in the lower v相似文献   

Probing the complete folding trajectory of a DNA hairpin using dual beam fluorescence fluctuation spectroscopy

The conformational fluctuations of dye-quencher labeled DNA hairpin molecules in aqueous solution were investigated using dual probe beam fluorescence fluctuation spectroscopy. The measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset optical probe regions, the absolute and relative concentrations of each conformational substate of the DNA, and the kinetics of the DNA hairpin folding and unfolding reactions in the 1 micros to 10 ms time range. A DNA hairpin containing a 21-nucleotide polythymine loop and a 4-base pair stem exhibited double exponential relaxation kinetics, with time constants of 84 and 393 micros. This confirms that folding and melting of the DNA hairpin structure is not a two state process but proceeds by way of metastable intermediate states. The fast time constant corresponds to formation and unfolding of an intermediate, and the slow time constant is due to formation and disruption of the fully base-paired stem. This is consistent with a previous study of a similar DNA hairpin with a 5-base pair stem, in which the fast reaction was attributed to the fluctuations of an intermediate DNA conformation [J. Am. Chem. Soc. 2006, 128, 1240-1249]. In that case, reactions involving the native conformation could not be observed directly due to the limited observation time range of the fluorescence correlation spectroscopy experiment. The intermediate states of the DNA hairpins are suggested to be due to a collapsed ensemble of folded hairpins containing various partially folded or misfolded conformations.     

Intermediate phases in the dodecyltrimethylammonium chloride/water system

Two intermediate phases have been found in the concentration range between the hexagonal and concentrated cubic phases in the binary system dodecyltrimethylammonium chloride (C₁₂TACl)/water. This region in the phase diagram was studied by means of ²H NMR of specifically deuteriated surfactant as well as by ¹⁴N NMR. Below 35°C, an intermediate phase with non-cylindrical aggregates is formed in the concentration range 80 to 84 wt% surfactant, X-ray data from this phase can be indexed to a centred rectangular lattice. In addition, there is a uniaxial phase with a reduced quadrupole splitting. The aggregates comprising the centred rectangular phase were analysed by means of bandshape analysis of the NMR spectra and by small angle X-ray scattering.     

Synthesis of 5-bromopyridyl-2-magnesium chloride and its application in the synthesis of functionalized pyridines

[reaction: see text] The 5-bromopyridyl-2-magnesium chloride (2), which was not accessible previously, was efficiently synthesized for the first time via an iodo-magnesium exchange reaction with 5-bromo-2-iodopyridine (1). This reactive intermediate was allowed to react with a variety of electrophiles to afford a range of useful functionalized pyridine derivatives. Application of this methodology to 5-bromo-2-iodo-3-picoline provided a simple and economical synthesis of a key intermediate for the preparation of Lonafarnib, a potent anticancer agent.     

Green synthesis of dihydroxybenzene from phenol with hydrogen peroxide catalyzed by iron modified FSM-16

Research on Chemical Intermediates - Catechol and hydroquinone are very important fine chemical intermediate products, which have a wide range of applications. The hydroxylation of phenol with...     

Insights into the Crystallization and Structural Evolution of Glycine Dihydrate by In Situ Solid‐State NMR Spectroscopy

In situ solid‐state NMR spectroscopy is exploited to monitor the structural evolution of a glycine/water glass phase formed on flash cooling an aqueous solution of glycine, with a range of modern solid‐state NMR methods applied to elucidate structural properties of the solid phases present. The glycine/water glass is shown to crystallize into an intermediate phase, which then transforms to the β polymorph of glycine. Our in situ NMR results fully corroborate the identity of the intermediate crystalline phase as glycine dihydrate, which was first proposed only very recently.     

Investigation of a flexible enantiospecific approach to aziridines

A flexible approach to functionalized and enantioenriched aziridines has been developed from an intermediate aziridinylmethyl tosylate. This protocol features a Cu-catalyzed Grignard substitution reaction that allows a range of functionalized organic fragments to be incorporated. Moreover, a convenient one-pot procedure is outlined that allows the trityl group to be exchanged for a range of common N-protecting groups.     

Microstructure of beta-lactoglobulin/pectin coacervates studied by small-angle neutron scattering

Small-angle neutron scattering (SANS) has been used to investigate the microstructure of beta-lactoglobulin/pectin coacervates prepared by different initial protein/polysaccharide weight ratio (r), sodium chloride concentration (C(NaCl)), and pectin charge density. The higher r and higher pectin charge density lead to higher scattering intensity at small q range (0.007 Angstrom(-1) < q < 0.02 Angstrom(-1)), suggesting that the charges of pectin chains are screened significantly by the binding of oppositely charged protein molecules, leading to a tighter aggregation of pectin chains. On the other hand, the appearance of a shoulder peak at intermediate q range (0.04 Angstrom(-1) < q < 0.2 Angstrom(-1)) is used to interpret the formation of protein domains in beta-lactoglobulin/pectin coacervates. At C(NaCl) = 0.1 M, the coacervate of beta-lactoglobulin and pectin A does not show a shoulder peak at intermediate q range at r = 10:1, suggesting that protein molecules are separately bound on pectin chains. However, a shoulder peak appears at intermediate q range at r = 20:1 and 30:1, and the average protein domain size estimated from the shoulder peak position is 7.2 and 8.5 nm, respectively, for these two coacervates. When C(NaCl) increases from 0.05 to 0.2 M, the shoulder peak shifts toward smaller q and becomes broader, indicating that the addition of a higher amount of salt leads to a more heterogeneous coacervate structure. Pectin B with a lower linear charge density favors the formation of larger protein domains. The formation of protein domains in beta-lactoglobulin/pectin coacervates is partially ascribed to the self-aggregation of beta-lactoglobulin molecules. Two kinds of microstructures of beta-lactoglobulin/pectin coacervates with and without observable protein domains have been proposed.     

Reaction of N-monosubstituted hydrazones with nitroolefins: a novel regioselective pyrazole synthesis

A novel regioselective synthesis of substituted pyrazoles from N-monosubstituted hydrazones and nitroolefins is described. The reaction is performed in a one-pot manner and the yields range from moderate to excellent. A key nitropyrazolidine intermediate is characterized and a plausible mechanism is proposed.     

Kinetics and mechanism of the NCN + NO2 reaction studied by experiment and theory

The rate constant for the NCN + NO 2 reaction has been measured by a laser photolysis/laser-induced fluorescence technique in the temperature range of 260-296 K at pressures between 100 and 500 Torr with He and N 2 as buffer gases. The NCN radical was produced from the photolysis of NCN 3 at 193 nm and monitored by laser-induced fluorescence with a dye laser at 329.01 nm. The rate constant was found to increase with pressure but decrease with temperature, indicating that the reaction occurs via a long-lived intermediate stabilized by collisions with buffer gas. The reaction mechanism and rate constant are also theoretically predicted for the temperature range of 200-2000 K and the He and N 2 pressure range of 10 (-4) Torr to 1000 atm based on dual-channel Rice-Ramsperger-Kassel-Marcus (RRKM) theory with the potential energy surface evaluated at the G2M//B3LYP/6-311+G(d) level. In the low-temperature range (<700 K), the most favorable reaction is the barrierless association channel that leads to the intermediate complex (NCN-NO 2). At high temperature, the direct O-abstraction reaction with a barrier of 9.8 kcal/mol becomes the dominant channel. The rate constant calculated by RRKM theory agrees reasonably well with experimental data.     

An improved procedure for the preparation of the β-hydroxy-α-alkyl fatty acid fragment of mycolic acids

An approach to a β-hydroxy-α-alkyl fatty acid intermediate that can be applied in the synthesis of a range of mycolic acids is described.     

Conformational changes of the S2YZ* intermediate of the S2 to S3 transition in photosystem II

The paper extends earlier studies on the S(2)Y(Z)* intermediate that is trapped by illumination in the temperature range 77 K to 190 K of untreated samples poised in the S(2)...Q(A) state. X-band EPR experiments on untreated and glycerol (50% v/v) treated samples at 10 K indicate that the intermediate consists of two components. A wide one with a splitting of ca 170 G, and a narrow one characterized by a splitting of ca 120 G (untreated), or 124 G (glycerol-treated samples). Lower temperatures of illumination in the above temperature range favor the wide component, which at 10 K decays faster than the narrow one. Re-illumination at 10 K after decay of the signal trapped at 77-190 K induces only the narrow component. Rapid scan experiments in the temperature range 77-190 K reveal high resolution spectra of the isolated tyz Z* radical and no evidence of alternative radicals. The two split signals are accordingly assigned to different conformations of the S(2)Y(Z)* intermediate A point-dipole simulation of the spectra yields "effective distances" between the spin densities of Y(Z)* and the Mn(4)Ca center of 5.7 ? for the wide and 6.4 ? for the narrow component. The results are discussed on the basis of a molecular model assuming two sequential proton transfers during oxidation of tyr Z. The wide component is assigned to a transient S(2)Y(Z)* conformation, that forms during the primary proton transfer.     

Pathway to copolymer collapse in dilute solution: uniform versus random distribution of comonomers

Monte Carlo simulations show that copolymers with uniformly (or periodically) distributed sticky comonomers collapse "cooperatively," abruptly forming a compact intermediate comprising a monomer shell surrounding a core of the aggregated comonomers. In comparison, random copolymers collapse through a relatively less-compact intermediate comprising a comonomer core surrounded by a fluffy monomer shell that densifies over a wide temperature range. This difference between the collapse pathways for random and uniform copolymers persists to higher chain lengths, where uniform copolymers tend to form multiple comonomer cores. In this paper, we describe the formation of such an intermediate state, and the subsequent collapse, by recognizing that these arise from the expected balance between comonomer aggregation enthalpy and loop formation entropy dictated by the chain microstructure.     

Pulsed‐EPR Evidence of a Manganese(II) Hydroxycarbonyl Intermediate in the Electrocatalytic Reduction of Carbon Dioxide by a Manganese Bipyridyl Derivative

A key intermediate in the electroconversion of carbon dioxide to carbon monoxide, catalyzed by a manganese tris(carbonyl) complex, is characterized. Different catalytic pathways and their potential reaction mechanisms are investigated using a large range of experimental and computational techniques. Sophisticated spectroscopic methods including UV/Vis absorption and pulsed‐EPR techniques (2P‐ESEEM and HYSCORE) were combined together with DFT calculations to successfully identify a key intermediate in the catalytic cycle of CO₂ reduction. The results directly show the formation of a metal–carboxylic acid–CO₂ adduct after oxidative addition of CO₂ and H⁺ to a Mn⁰ carbonyl dimer, an unexpected intermediate.